Project Summary/Abstract Using the unique resources of the ALIVE cohort of persons who inject drugs (PWID) led by study co- investigator Gregory Kirk and the extensive experience in reservoir biology provided by co-investigators Rafick Sekaly, Joel Blankson, Greg Laird, and study PI Robert Siliciano, we will investigate the effects of heroin and cocaine use on the latent reservoir for HIV-1 in CD4+ T cells, which is the major barrier to cure. We hypothesize that by affecting inflammatory and immune pathways, active heroin and/or cocaine may alter T cell homeostasis and the distribution of HIV-1 proviruses among memory cells subsets, thereby affecting the size, turnover, and inducibility of the latent reservoir. In addition, we hypothesize that past injection drug use and the associated unstructured treatment interruptions will permanently affect the size and inducibility of the latent reservoir. Active drug use and legacy effects of past drug use may also affect that ability of host effector cells to clear infected cells during curative interventions. These hypotheses will be tested using samples from ALIVE cohort participants and experimental approaches informed by full genome proviral sequencing in order to achieve a better understanding of the reservoir in PWID and inform how curative strategies can be applied to this population. We will first determine the effects of active heroin/cocaine use on the latent reservoir by quantifying the size of the reservoir in resting CD4+ T cells and T cell subsets in active drug users with a novel high-throughput assay for intact proviruses. This assay is based on full genome sequence analysis by the Siliciano lab that led to the discovery that most proviruses (98%) are defective. Thus we will use a novel digital droplet PCR assay that selectively detects proviruses that are intact and have the potential to cause viral rebound. We will also measure the inducibility of latent proviruses in response to T cell activation and latency reversing agents using the TILDA assay developed by Dr. Sekaly and colleages. In addition, we will carry out full genome sequencing of HIV-1 proviruses to define additional properties that are relevant to HIV-1 cure including replication capacity, clonality, turnover rates, and escape mutations in cytolytic T lymphocyte (CTL) epitopes. These studies will be carried out in PWID who are actively using as well as those who are no longer using in order to distinguish active and legacy effects of drug use. We will also measure the ability of autologous CD8+ CTL and NK cells from active drug users to eliminate autologous infected cells following latency reversal and carry out similar studies in PWID who are no longer actively using to distinguish direct drug effects from legacy effects of drug past drug use on effector cell function. Together these studies of the size, inducibility, and composition of the reservoir, and the ability of host effector cells to clear infected cells, should provide critical information for the design of cure strategies for PWID and guide longitudinal and mechanistic studies in the second phase of the grant.